KAR9 Is a Novel Gene
In a collection of karyogamy mutants defective for nuclear congression, kar9
was unique in exhibiting misoriented cytoplasmic microtubules in the zygote (Kurihara et al., 1994
). In addition, we found that the kar9
mutants were sensitive to the microtubule-destabilizing drug, benomyl. Growth was significantly suppressed at 15 μg/ml at 30°C (data not shown). In contrast, wild type was resistant to 25 μg/ml benomyl. As described below, kar9
mutants also showed mitotic defects consistent with a defect in nuclear migration. The karyogamy defect, the benomyl sensitivity, and the mitotic defect segregated together in five tetrads derived from meiotic crosses. Therefore, we considered it highly likely that each of these phenotypes was the result of the kar9
mutation. These phenotypes also suggested that Kar9p might play a broader role in microtubule function. To investigate further, the wild-type KAR9
gene was isolated by suppression of kar9'
s benomyl sensitivity. A kar9-485
strain (MS2684) was transformed with a YCp-based yeast genomic library (Rose et al., 1987
) and screened for plasmid-dependent benomyl resistance (20 μg/ml at 30°C). Three independent plasmids were recovered that suppressed both the benomyl sensitivity and mitotic defects of kar9-485.
These plasmids contained inserts that overlapped by 5.8 kb (Fig.1). To identify the minimal complementing region, additional subclones were constructed and tested for their ability to complement kar9-485'
s benomyl sensitivity and mitotic defects. Subclones containing all or almost all of the KAR9
ORF corrected the benomyl sensitivity defect (Fig. ). In contrast, two subclones truncated at the NH2
terminus only partially suppressed the mitotic defect.
To determine that the region on the three overlapping plasmid inserts contained the authentic KAR9 gene rather than an extragenic suppressor, the 2.1-kb SpeI/SpeI fragment within this region was used for linkage analysis (Fig. ). Integration of this fragment on pMR2886 was used to mark the wild-type KAR9 locus with URA3. When crossed to a kar9 mutant, the marked allele segregated together with the benomyl resistance in 31 out of 32 tetrads analyzed (see Materials and Methods). These results indicate that the sequences contained on pMR2886 were tightly linked to and therefore derived from the authentic KAR9 gene.
Hybridization methods demonstrated that KAR9
mapped to the distal end of the left arm of chromosome XVI. DNA sequence analysis showed that KAR9
was between MDL2
(Fig. ). This sequence was subsequently confirmed by the S. cerevisiae
genome sequencing project. KAR9
potentially encoded a 644–amino acid, 74-kD protein (Fig. ) that showed no significant homology to any known proteins in available databases using BLAST or FASTA programs (Pearson and Lipman, 1988
). Kar9p is predicted to be a basic protein with an overall calculated average isoelectric point (pI) of 8.9. However, the NH2
-terminal 175 amino acids is predicted to be very acidic with an average calculated pI of 4.1. The COOH half of the protein contains three regions, which are predicted to be very basic, each with a calculated pI > 11.4 (Fig. ). This bipolar distribution of electrostatic charges is similar to that of the microtubule-associated protein, MAPU (Aizawa et al., 1990
). The COOH-terminal third of the protein is also predicted to be proline rich, containing ~10% proline residues and three PXXP motifs, possibly indicating an interaction with SH3 proteins (Yu et al., 1994
; Alexandropoulos et al., 1995
). Alternatively, basic and proline-rich regions are also characteristic of microtubule-binding proteins (Meluh and Rose, 1990
). However, the basic domain of Kar9p does not have sequence homology to other known microtubule-binding proteins, including TAU, MAPU, Kar3p, Mhp1p, Bik1p, and p150glued
. Nevertheless, the possibility that Kar9p may possess a microtubule-binding domain is supported by the behavior of GFP-Kar9p described below. Together, these data indicate that KAR9
is a novel gene.
Figure 2 Kar9p sequence features. (A) Structural representation of Kar9p domains. Vertical-striped region represents the acidic domain of Kar9p. The horizontal-striped region represents the putative coiled-coil. The diagonally striped areas represent the highly (more ...)
KAR9 Is Not Essential for Life, but Is Required for Cytoplasmic Microtubule Orientation and Nuclear Migration
To determine if the KAR9
gene product performs an essential function, we replaced the KAR9
sequence encoding amino acids 7–644 with LEU2
in haploid cells by homologous recombination using the one-step gene replacement method (Rothstein, 1983
). The recovery of viable haploid colonies demonstrated that KAR9
is not essential for life. As described below, the kar9Δ
mutants exhibited the same array of phenotypes as the kar9-485
point mutant, confirming the identification of the authentic KAR9
gene. Given the defects in cytoplasmic microtubule orientation in zygotes and the benomyl sensitivity of mitotic cells, we next examined the nuclear migration and microtubule morphology of the kar9Δ
mutant in detail.
To determine how early in the mating process the defects were evident, we examined nuclear positioning and microtubule orientation in kar9Δ
mutant shmoos. First, kar9Δ
mutants were arrested with α factor and scored for nuclear position using DAPI stain. In wild-type shmoos, the nucleus normally moves up to the neck of the pear-shaped shmoo in preparation for mating (Byers and Goetsch, 1975
; Rose, 1991
; Read et al., 1992
). Confirming this observation, we found that in 93% of wild-type shmoos with projections (n
= 401), the nuclei were in or at the neck of the shmoo (Table ). Like wild type, the nuclei of dynein mutants were positioned correctly in 94% of shmoos (n
= 400). In contrast, the nuclei of kar9Δ
mutants were found in the center of the cell in ~60% of shmoos (n
= 300 and n
= 324). We next used indirect immunofluorescence microscopy to examine the microtubules in the shmoo. In wild-type shmoos, 85% or more of the cells had microtubule bundles that extended to the shmoo tip (Fig. A
). In contrast, 73% of the kar9Δ
mutant shmoos had misoriented microtubule bundles that did not extend into the shmoo tip (Fig. D
). Thus, the mating defect of the kar9
mutant is likely to be the result not only of the failure of cytoplasmic microtubules within the zygote to interdigitate, but also of an earlier defect in which cytoplasmic microtubules were misoriented in the shmoo.
Nuclear Migration Defect in kar9Δ Shmoos
Figure 3 kar9 defects: aberrant cytoplasmic microtubules and nuclear migration. Wild-type (A–C) (MS1556) and kar9Δ (D–F) (MS4306) shmoos, as well as kar9-485 vegetatively growing cells (G–I) (MS2684) were fixed for indirect immunofluorescence (more ...)
mutants are sensitive to benomyl and because microtubule orientation is important during mitosis, we examined the kar9
mutants for nuclear positioning defects in mitosis. First, cultures of asynchronously growing kar9
cultures were stained with DAPI and scored for defects in nuclear location (Fig. , J–L
mutants exhibited significantly increased frequencies of abnormal mitotic phenotypes in large-budded cells. These included cells that contained a single nucleus that had failed to migrate to the bud neck (Fig. J
), cells with mitosis occurring within the mother cell (Fig. K
), and cells with two nuclei within the mother cell (Fig. L
). Anucleate cells were rarely seen (<0.5%) in kar9
mutant cultures. Cells exhibiting these three defects usually totaled between 10 and 15% of the culture at 30°C (Table ). In contrast, wild-type cells showed only 2–3% of such abnormal cells. Because similar defects were reported for dhc1
mutants (Eshel et al., 1993
; Li et al., 1993
), we compared directly the nuclear migration defects observed in dhc1
with those of kar9
(Table ). While the dhc1
mutant phenotypes were less severe at 30°C, the magnitude of the defects for both strains was aggravated by growth in the cold, with the sum of all defects increasing to >20% at 12°C. Thus, both dhc1
exhibited strikingly similar defects in nuclear positioning (Table ).
Nuclear Positioning Defects in kar9 Mutants
Indirect immunofluorescence was carried out to determine if the kar9Δ
mutant exhibited mitotic nuclear migration defects that correlated with microtubule abnormalities. Exponentially growing cultures of kar9
mutants contained many cells with abnormal microtubule morphologies for both the mitotic spindle and the cytoplasmic microtubules (Fig. G
). The mitotic spindle was often rotated away from the long axis of the mother–bud. Occasionally, the spindle appeared bent along the inner surface of the mother cell (data not shown), similar to that observed in other nuclear migration mutants, DHC1
(Eshel et al., 1993
; Li et al., 1993
(Clark and Meyer, 1994
; Muhua et al., 1994
), and JNM1
(McMillan and Tatchell, 1994
). The cytoplasmic microtubules were also frequently positioned aberrantly in kar9
mutants (Fig. G
). In contrast to both wild-type and dhc1Δ
cells, 30% of kar9Δ
cells carrying out anaphase within the mother cell had cytoplasmic microtubules that failed to extend into the bud (Fig. A
). In those kar9Δ
cells that contained two nuclei within the mother cell, 70% exhibited cytoplasmic microtubules that did not extend into the bud (Fig. B
). This is in sharp contrast to the phenotype of equivalent dhc1Δ
mutant cells, in which 83% of the cytoplasmic microtubule bundles were observed to extend into the bud (Fig. B
; Li et al., 1993
Figure 4 Quantitation of cytoplasmic microtubule orientation defect. The percentage of cells with cytoplasmic microtubule orientation defects was determined in A (cells carrying out anaphase within the mother cell), and B (cells with two nuclei within the mother (more ...)
The kar9Δ mutants displayed no obvious actin defect by rhodamine-phalloidin staining. In kar9, actin patches localized to the growing bud in small-budded cells and to the cleavage furrow in cells undergoing cytokinesis as they normally do in wild type (data not shown). From this, together with the benomyl sensitivity, we conclude that the kar9 mutation affects the functions of cytoplasmic microtubules.
kar9Δ and dhc1Δ Mutants Are Synthetically Lethal
Because kar9 and dhc1 display similar phenotypes in nuclear migration, we wanted to determine whether KAR9 functions in the same or different pathways as other genes involved in this process. To carry out the analysis, marked deletions of KAR9 and several other nuclear migration genes were crossed together.
The first nuclear migration protein tested was the microtubule motor protein, dynein, encoded by DHC1/DYN1. To make the kar9Δ dhc1Δ mutant, the dhc1Δ strain (MS4262) was crossed to kar9-Δ1::LEU2 (MS4589). Of the 35 segregants predicted to be double mutants on the basis of segregation of markers in sister spores, 9% failed to germinate and 91% formed microcolonies (Table ; Fig. , A and B) at 30°C. In contrast, wild-type and single mutant colonies showed 97% spore viability and no apparent growth defects (data not shown). The microcolonies exhibited heterogeneity in size, but by 3–4 d after germination at 30°C they usually attained the size of an 18–24-h-old wild-type colony (e.g., <1,000 cells). Examination of the cells within these microcolonies by DAPI staining and Nomarski optics revealed a wide range of defects. Most cells were multinucleate, anucleate, deformed in cell shape, or lysed. These defects are consistent with a severe defect in nuclear migration (Fig. B).
Viability of kar9Δ Double Mutants
Figure 5 Synthetic lethality. kar9-Δ1::LEU2 (MS4589) was crossed to dhc1Δ::URA (MS4262). Spores were allowed to germinate 4 d on YPD and photographed (A). Arrows show double-mutant microcolonies. 15–20 microcolonies were removed from (more ...)
Several genes, including ACT5
, have been suggested to operate in nuclear migration in concert with the dynein heavy chain as part of the dynactin complex (McMillan and Tatchell, 1994
; Muhua et al., 1994
). If such a model is correct, then deletions in these genes would be predicted also to result in synthetic lethality when combined with the KAR9
deletion. To test this possibility, the jnmΔ
strain (MS4321) was crossed to the kar9-Δ2::HIS3
strain (MS4587) to create kar9Δ jnm1Δ
mutants. Seventeen tetrads were examined, yielding 14 predicted double mutants. Of these, 100% formed microcolonies (Table ). To create kar9Δ act5Δ
double mutants, the ACT5
delete strain (MS4586) was crossed to the kar9-Δ1::LEU2
strain (MS4263). From this cross, 23 tetrads were dissected, producing 20 predicted double mutants. Of these double mutants, 5% were dead and 95% formed microcolonies (Table ). Like the kar9Δ dhc1Δ
double mutant, analysis of the cells within these microcolonies also revealed lysed cells, anucleate cells, and multinucleate cells. In contrast, dhc1 act5
, dhc1 jnm1
, and act5 jnm1
double mutants are viable and show no more severe defect than any single mutant (Tatchell, K., personal communication; Muhua et al., 1994
; Geiser et al., 1997
). We conclude therefore that Kar9p acts in a nuclear migration pathway that is separate from and partially redundant with that of the dynein/dynactin complex.
Several additional genes were also tested for the possibility of genetic interactions with kar9Δ
. A bilateral karyogamy mutation, bik1
(Berlin et al., 1990
), with additional functions during mitosis, also exhibited a microcolony phenotype in combination with KAR9
deletions (Table ). Cells found within the microcolonies of the kar9Δ bik1-518
(Trueheart et al., 1987
) double mutant (Fig. C
) exhibited defects similar to those of the kar9Δ dhc1Δ
To test the specificity of the synthetic lethality, crosses to mutants in other microtubule motor proteins were performed. KIP1
(Roof et al., 1992
) and CIN8
(Hoyt et al., 1992
) are required for establishment of a bipolar mitotic spindle. kar9Δ cin8Δ
and kar9Δ kip1Δ
double mutants resulted in no detectable growth defect (Table ). KAR3
is required for mitosis and mating, possibly by mediating the sliding of microtubules past each other (Meluh and Rose, 1990
). The kar9Δ kar3Δ
mutant spores exhibited no obvious growth defect worse than that of the kar3Δ
single mutant alone. Deletions in SMY1
, a kinesin involved in secretion (Lillie and Brown, 1992
), also exhibited no apparent growth defect when combined with the KAR9
deletion (Table ).
GFP-Kar9p Localizes to the Tip of the Shmoo
To better understand how Kar9p might be functioning, its localization was determined inside living yeast cells. A GFP-Kar9p fusion protein was constructed on a centromere-based plasmid with its expression under the control of the GAL1 promoter (pMR3465). This construct fully suppressed the microtubule orientation and nuclear migration defects in kar9Δ shmoos (data not shown). The localization of GFP-Kar9p was examined in shmoos and zygotes. In each instance, GFP-Kar9p fluorescence was observed primarily as a single small dot. In 73% of kar9Δ shmoos, the dot was located at the tip of the shmoo projection (n = 152) (Fig. A). In an additional 12% of the shmoos, a thin line extended from the single dot toward the nucleus (data not shown). Identical results were observed in wild-type cells expressing GFP-Kar9p. In zygotes, GFP-Kar9p was located as an elongated dot at the future site of cell fusion of the pre-zygote (100%; n = 15) (Fig. D), as if two dots had fused. In budded zygotes, it was also found at the tip of the emerging bud (Fig. G).
Figure 6 Localization of GFP-Kar9p in shmoos and zygotes. kar9Δ shmoos (MS4382) (A–C), pre-zygotes (MS4671X MS4387) (D–F), and budding zygotes (MS4671X MS4387) (G–I), each expressing GFP-Kar9p (pMR3465) were fixed and stained (more ...)
GFP-Kar9p Localization to the Tip of the Bud Is Cell-Cycle Dependent
To gain an understanding of how Kar9p might be functioning to orient microtubules in mitotically growing cells, the localization of GFP-Kar9p was scored throughout the cell cycle. First, asynchronously growing cultures were examined using bud size and nuclear position as an indicator of the cell cycle stage. In unbudded cells, little or no GFP localization was observed (76%; n = 51) (Figs. A and ). In small–medium-budded cells, the majority of cells showed GFP-Kar9p localization as a single dot at the tip of the growing bud (58%; n = 57) (Figs. D and ). An additional 14% of small-budded to medium-budded cells also had the dot at the tip of the bud with a second dot between the first cortical dot and the nucleus (Fig. ). At anaphase, the major localization pattern (49% of all anaphase cells; n = 57) was a single dot of GFP at the tip of the bud (Figs. G and ). An additional 14% had the dot at the tip of the bud, but also had a second dot between the cortical dot and the nucleus (Fig. ). For the small-budded and anaphase stages, the percentage of cells that showed no localization was relatively low, 19 and 30%, respectively (Fig. ). However at telophase, the majority of cells exhibited no localization (58%; n = 80) (Figs. J and ). To confirm the latter stages of localization, cells were first synchronized with hydroxyurea, and then released from the block. Under these conditions, >85% of telophase cells showed no localization (n = 70). In comparison, only 17% of anaphase cells exhibited no localization at the corresponding time point (n = 88) (Fig. ). In addition to the single dot in the bud and the “two dots in a line” patterns (Fig. ), some cells also exhibited an additional spot. These cells were classified as “other” (Fig. ). The additional spot was located at one of the six locations depicted in Fig. M in >90% of cells in the “other” category. In a few rare examples, three spots per cell were observed. We conclude that GFP-Kar9p shows both mother–daughter asymmetry and cell cycle dependence for its localization.
Figure 7 Cell cycle–dependent localization of GFP-Kar9p. The cell cycle position of a wild-type strain expressing GFP-Kar9p (MS4387) was estimated based on bud size and nuclear morphology. The GFP localization pattern was then determined in unbudded (more ...)
Figure 8 Quantification of GFP-Kar9p localization. The cell cycle position of cells expressing GFP-Kar9p was estimated as described in Fig. . Cells in the Other category had one or more dots in the locations depicted in Fig. M. Subsets (more ...)
Cytoplasmic Microtubules Intersect the GFP-Kar9p Dot
To determine the relationship between cytoplasmic microtubules and the GFP-Kar9p dot, double-label indirect immunofluorescence was conducted using antibodies specific for tubulin and GFP. In shmoos, the cytoplasmic microtubule bundle terminated at the dot of anti-GFP staining in all cases (n = 100) (Fig. , A–D). In vegetative cells, microtubule staining intersected the anti-GFP staining dot in 85% of the large-budded cells examined (n = 20; Fig. , E–H). In a small percentage of both shmoos and large-budded cells, a line of anti-GFP immunofluorescence extended away from the anti-GFP dot toward the nucleus. In all cases, the line of staining colocalized with the cytoplasmic microtubules.
Figure 9 Cytoplasmic microtubules intersect the GFP-Kar9p dot. Double-label indirect immunofluorescence was carried out on wild-type shmoos (A–D) and cells (E–H) expressing GFP-Kar9p using anti-GFP and anti-tubulin (YOL 1/34). Two shmoos are (more ...)
GFP-Kar9p Localization at the Cortex Is Independent of Microtubules
Kar9p might function in microtubule orientation by two different mechanisms. In the first model, Kar9p might function as a microtubule-associated protein which stabilizes cytoplasmic microtubules. Localization of Kar9p at the cortex would therefore be dependent solely on the cytoplasmic microtubules. Alternatively, Kar9p might instead serve as a target for the cytoplasmic microtubules at the cortex. In this second model, Kar9p localization would be solely dependent on cortical information and independent of microtubules.
To examine these possibilities and to ascertain whether microtubules are required for the maintenance of Kar9p localization, wild-type shmoos already expressing GFP-Kar9p were treated with nocodazole to depolymerize preexisting microtubules (Fig. B) or mock-treated with DMSO (Fig. A). 74% of shmoos treated with nocodazole under these conditions had a GFP-Kar9p dot at the tip of the shmoo, as compared to 90% of the mock-treated control shmoos. Thus, Kar9p remained at the tip of these shmoos and microtubules were not required to maintain GFP-Kar9p at its cortical location. Alternatively, microtubules might play a role in establishing the localization of GFP-Kar9p at its cortical site. To investigate this possibility, cells were induced to form shmoos either in the presence (Fig. C) or absence of microtubules (Fig. D) and the expression of GFP-Kar9p was then induced. GFP-Kar9p localization was found at the tip of 72% of shmoos treated with nocodazole versus 85% of shmoos mock treated with DMSO. Thus, under microtubule-depolymerizing conditions, GFP-Kar9p was still able to localize to the tip of the shmoo. Identical results were obtained using the GFP-KAR9 plasmid in a kar9 deletion strain.
Figure 10 Kar9p localization at the cortex is independent of microtubules. The wild-type strain (MS1556) containing the GFP-KAR9 plasmid (pMR3465), was grown to early exponential phase. Representative examples of each phenotype scored are shown. 100 cells were (more ...)
A similar analysis was carried out to determine if GFP-Kar9p localization was also independent of microtubules in vegetative cells. When microtubules were depolymerized after induction of GFP-Kar9p, localization was found at the tip of the bud in 89% of large-budded cells (Fig. F). In comparison, in the mock-treated controls, 72% had the GFP-Kar9p dot at the tip of the large bud (Fig. E). Similarly, when microtubules were depolymerized before the induction of GFP-Kar9p, 79% of large-budded cells contained a dot of GFP-Kar9p localization at the tip of the bud (Fig. H). Thus, without microtubules present, GFP-Kar9p could still localize at the tip of the bud. Therefore, we conclude that like shmoos, Kar9p localization at the cortex was independent of microtubules in vegetative cells.
In 11 and 12% of the mock-treated control cells, a line of GFP-Kar9p localization extending from the cortex was also observed (Fig. , E and G). When cells were treated with nocodazole, none of the cells displayed the line of GFP-Kar9p fluorescence (Fig. , F and H). In the experiment with shmoos, a GFP-Kar9p dot with a line of fluorescence was scored in the “cortical dot” category. When the shmoos were treated with nocodazole, the lines of GFP-Kar9p fluorescence were not observed (data not shown). Therefore, localization along microtubules was dependent on their polymerization. These data support the finding that the line of GFP-Kar9p fluorescence colocalized with microtubules (Fig. ) and are consistent with the idea that Kar9p may contain a microtubule-binding domain.